Author(s):Taqdees Malik,
Department of Microbiology, University of Karachi, Karachi, PakistanAsma Naim*,
Department of Microbiology, University of Karachi, Karachi, PakistanAsma Saeed.
Department of Microbiology, University of Karachi, Karachi, Pakistan

Graphical Abstract:

Abstract:

Introduction: In the past few years, the use of antimicrobial drugs against a wide range of
pathogens has increased significantly. This extensive use of drugs has increased the resistance rate in
microbial community widely. Molecular techniques for the detection of resistance are more reliable as
compared to the conventional phenotypic method.

Background: The family Enterobacteriaceae is considered to be an important cause of nosocomial infections
due to its predominantly active species such as E. coli, Klebsiella and Pseudomonas. These
organisms are mainly involved in causing pneumonia, sepsis, post-surgical and urinary tract infections.
Resistance against antimicrobial drugs among these isolates is increasing more rapidly all over the
world. This study primarily focuses on the resistant isolates of Klebsiella species. The drug resistance in
Klebsiella isolates is found to be associated with the production of resistance enzymes such as beta
lactamase and extended spectrum beta lactamase (ESBLs) which confer resistance, most specifically
against cephalosporins and extended spectrum cephalosporins. Since these enzymes are plasmid mediated,
they can also produce resistance against several other antimicrobials. It has been found that among
the genus Klebsiella, ESBLs are more prevalent in K. pneumoniae followed by K. oxytoca.

Methods: In this study, we estimated the distribution of ESBL producers among Klebsiella species and
performed their genetic characterization. A total of 236 gram-negative isolates were collected from
different microbiological laboratories, during the period January 2010 till January 2012. Among these
gram-negative isolates, 125 were identified as Klebsiella species. After species identification, Kirby
Bauer disk diffusion method was used for antimicrobial susceptibility profiling. Furthermore, the phenotypic
detection of ESBL producers was performed by double disc synergy and combination disc
methods. Resistance genes responsible for the production of beta lactamase and extended spectrum beta
lactamase enzymes were detected by Polymerase chain reaction. DNA sequencing was performed by
selecting an ESBL producing Klebsiella pneumoniae strain with a positive blaTEM gene.

Results: In this study, we found 48%, 43.2% and 2.4% of SHV, TEM, and CTX-M resistance genes
respectively in Klebsiella isolates. The DNA sequence ESBL and blaTEM positive Klebsiella strain
showed 94% similarity with Klebsiella pneumoniae KUN5033 blaTEM gene for class A beta-lactamase
TEM-198 analyzed by the Basic Local Alignment Search Tool (BLAST).

Conclusion: Since there is an intense need of research in the field of drug resistance for implementing
strict antibiotic control policies in hospitals, health care centers, laboratories, etc., the present study is
dedicatedly conducted to estimate the drug resistant Klebsiella isolates, specifically the Beta lactamase
and Extended Spectrum Beta Lactamase producers, at molecular level.

Current Drug Delivery

Affiliation:Department of Microbiology, University of Karachi, Karachi, Department of Microbiology, University of Karachi, Karachi, Department of Microbiology, University of Karachi, Karachi

Keywords:TEM, SHV, CTX-M, klebsiella, ESBL, drug-resistance.

Abstract:Introduction: In the past few years, the use of antimicrobial drugs against a wide range of
pathogens has increased significantly. This extensive use of drugs has increased the resistance rate in
microbial community widely. Molecular techniques for the detection of resistance are more reliable as
compared to the conventional phenotypic method.

Background: The family Enterobacteriaceae is considered to be an important cause of nosocomial infections
due to its predominantly active species such as E. coli, Klebsiella and Pseudomonas. These
organisms are mainly involved in causing pneumonia, sepsis, post-surgical and urinary tract infections.
Resistance against antimicrobial drugs among these isolates is increasing more rapidly all over the
world. This study primarily focuses on the resistant isolates of Klebsiella species. The drug resistance in
Klebsiella isolates is found to be associated with the production of resistance enzymes such as beta
lactamase and extended spectrum beta lactamase (ESBLs) which confer resistance, most specifically
against cephalosporins and extended spectrum cephalosporins. Since these enzymes are plasmid mediated,
they can also produce resistance against several other antimicrobials. It has been found that among
the genus Klebsiella, ESBLs are more prevalent in K. pneumoniae followed by K. oxytoca.

Methods: In this study, we estimated the distribution of ESBL producers among Klebsiella species and
performed their genetic characterization. A total of 236 gram-negative isolates were collected from
different microbiological laboratories, during the period January 2010 till January 2012. Among these
gram-negative isolates, 125 were identified as Klebsiella species. After species identification, Kirby
Bauer disk diffusion method was used for antimicrobial susceptibility profiling. Furthermore, the phenotypic
detection of ESBL producers was performed by double disc synergy and combination disc
methods. Resistance genes responsible for the production of beta lactamase and extended spectrum beta
lactamase enzymes were detected by Polymerase chain reaction. DNA sequencing was performed by
selecting an ESBL producing Klebsiella pneumoniae strain with a positive blaTEM gene.

Results: In this study, we found 48%, 43.2% and 2.4% of SHV, TEM, and CTX-M resistance genes
respectively in Klebsiella isolates. The DNA sequence ESBL and blaTEM positive Klebsiella strain
showed 94% similarity with Klebsiella pneumoniae KUN5033 blaTEM gene for class A beta-lactamase
TEM-198 analyzed by the Basic Local Alignment Search Tool (BLAST).

Conclusion: Since there is an intense need of research in the field of drug resistance for implementing
strict antibiotic control policies in hospitals, health care centers, laboratories, etc., the present study is
dedicatedly conducted to estimate the drug resistant Klebsiella isolates, specifically the Beta lactamase
and Extended Spectrum Beta Lactamase producers, at molecular level.